Method and device for the determination of platelet function under flow conditions

ABSTRACT

The invention lies in the area of platelet function diagnostics and relates to a method for the determination of platelet function under flow conditions as well as a device for the implementation of this method. The method is particularly suitable for the determination of the effect of clopidogrel and of other P2Y(12) antagonists with antithrombotic activity as well as the determination of P2Y(1) antagonists with antithrombotic activity.

This application claims the benefit of priority under 35 U.S.C. § 119 toGerman Patent Application No. DE 10 2006 020 385.2, filed on Apr. 28,2006, incorporated herein by reference.

The invention lies in the area of platelet function diagnostics andrelates to an in vitro method for the determination of platelet functionunder flow conditions as well as a device for the implementation of thismethod. The method is particularly suitable for the determination of theeffect of clopidogrel after oral intake and of other P2Y(12) antagonistswith antithrombotic activity. Furthermore, the blockade of the secondplatelet ADP receptor (P2Y(1) receptor) by specific antagonists can alsobe detected with the method.

Physiological processes that on the one hand guarantee the fluidity ofblood in the vascular system and on the other avoid extravascular bloodloss through the formation of blood clots are classified under the termhemostasis. Numerous protein factors are involved in the regulation ofhemostasis as well as also cellular components, for example thrombocytes(platelets). In the case of vessel damage attachment of platelets to thesubendothelial collagen first takes place. This adhesion is mediated byadhesion proteins such as the von Willebrand factor (VWF). During theadhesion process the platelets are activated and release mediators fromtheir granulae through which the aggregation of further platelets and anincrease in activation are induced. In this way primary vessel wallocclusion (primary hemostasis) takes place which then is furtherstabilized by reactions of the plasmatic coagulation system (secondaryhemostasis). Dysregulation of these processes can lead to thrombophiliaor a tendency towards hemorrhage, which dependent upon the degree ofseverity can have life-threatening consequences.

Different in vitro test methods have been developed in coagulationdiagnostics which help to determine whether the blood of a patientcoagulates properly or whether a coagulation defect is present. In thecase of a coagulation defect it is frequently necessary to obtainprecise information on the cause of the defect present in order to beable to select the optimal therapeutic measures. An importantsub-function of the coagulation system that can be investigatedspecifically is primary hemostasis, which is essentially dependent onthe functionality of the platelets.

Methods to determine platelet function are not only used for thediagnosis of acquired or inherited platelet dysfunction, but also formonitoring antithrombotic therapies. Medication that inhibits theaggregation of platelets is used mainly for the prophylaxis and therapyof arterial thromboembolitic events such as myocardial infarction orstroke. The most widely used active compounds with platelet aggregationinhibitory effect are acetylsalicylic acid (ASA) and the thienopyridinesclopidogrel and ticlopidine. ASA irreversibly inhibits cyclooxygenase-1(COX-1), an intracellular enzyme that is involved in the synthesis ofthe platelet aggregation promoter thromboxane A2. Owing to their mode ofaction clopidogrel and ticlopidine belong to the class of P2Y(12)antagonists. After oral intake of clopidogrel or ticlopidine metabolitesare formed in the liver that block selectively the purinergic P2Y(12)receptor. The purinergic P2Y(12) receptor is expressed on the plateletsurface and can be activated by extracellular adenosine-5′-diphosphate(ADP). As a consequence of the activation of the purinergic P2Y(12)receptor intracellular processes are induced in the platelets, forexample the inhibition of the formation of cAMP, that give rise to aplatelet aggregation reaction. P2Y(12) antagonists block the purinergicP2Y(12) receptors on the platelet surface and thus possess anantithrombotic activity.

The second purinergic ADP receptor P2Y(1) is also expressed on theplatelet surface and is activated by extracellularadenosine-5′-diphosphate. As a consequence of the activation of thepurinergic P2Y(1) receptor intracellular processes are initiated in theplatelets, for example an increase in intracellular calcium, that giverise to a platelet aggregation reaction. P2Y(1) receptor antagonists actagainst this process and thus have an antithrombotic activity.

Precise knowledge of the status of the platelet function of patients whoare receiving antithrombotic therapy is considered to be increasinglyimportant since, for example, the occurrence of so-called clopidogrelresistance is under serious consideration as an increasing risk factor.Clopidogrel resistance is present when the platelet function of apatient is only slightly influenced by the administration of a standarddose of clopidogrel, or not at all. On the one hand a test can becarried out to determine whether an adequate antithrombotic response isactually achieved with a selected dose by determination of plateletfunction. On the other hand, doses or responses of an antithromboticmedication that are too high can be determined and treated, which isnecessary, for example, prior to surgery in order to exclude bleedingcomplications.

Different methods for the investigation of platelet function are knownin the prior art. Bleeding time determination is a global in vivo testwhich records primary hemostasis. The bleeding time is determinedwherein the patient is given a small cut or prick injury and the timefor coagulation is measured. It is a poorly standardizable, coarselyinformative test that is used primarily in an emergency situation inorder to obtain an overview of primary hemostasis. Taking plateletaggregation inhibitors leads to an increase in bleeding time. Thedisadvantage of bleeding time determination is that platelet dysfunctioncannot be excluded even with a normal bleeding time.

Different in vitro methods allow a significantly more sensitivedetection of platelet dysfunction. Normally in these methods plateletaggregation is induced in a whole blood sample or in a sample ofplatelet-rich plasma (PRP) by the addition of an activator and theaggregation reaction is measured. The most commonly used activators usedfor the induction of platelet activation are ADP(adenosine-5′-diphosphate), collagen, epinephrine (adrenaline),ristocetin and different combinations thereof as well as thrombin, TRAP(thrombin receptor activating protein) or serotonin.

In light transmission aggregometry, also known as Born plateletaggregation, the aggregation efficiency of platelets in platelet-richplasma is measured photometrically in the presence ofaggregation-inducing compounds in an aggregometer. The lighttransmission of the PRP sample is increased due to aggregate formationso that the rate of aggregate formation, for example, can be determinedby measurement of light transmission. The therapeutic effects ofplatelet aggregation inhibitors used medically can also be determinedwith the aid of light transmission aggregometry. A disadvantage of lighttransmission aggregometry is that only platelet-rich plasma can be usedas sample material. Platelet-rich plasma lacks not only important bloodcomponents such as, for example, red and white blood cells, but alsorequires a time-consuming and error-prone sample preparation.

Another test principle for the determination of platelet function isrealized in the Platelet Function Analyzer (PFA-100®, Dade BehringMarburg GmbH, Marburg, Germany). The PFA-100® is a global, automated andstandardized in vitro whole blood test with which primary hemostasis ismeasured under flow conditions and thus in the presence of high shearforces. In order to simulate the flow conditions and the shear forcesthat prevail in the smaller arterial blood vessels a partial vacuum ofabout −40 mbar is produced in a special test cartridge. The citratedwhole blood that is located in a sample reservoir is sucked through acapillary with a diameter of about 200 μm. The capillary leads into ameasurement chamber which is closed with a partition member, for examplea membrane, which has a central capillary opening (aperture) throughwhich the blood flows due to the partial vacuum. In most cases themembrane, at least within the region surrounding the aperture, is coatedwith one or more activators that induce platelet aggregation so that thepassing blood comes into contact with the aggregation-inducingsubstances in the region of the aperture. As a consequence of theinduced adhesion and aggregation of the platelets a thrombus is formedin the region of the aperture which seals the membrane opening and stopsthe blood flow. In this system the time required to close the membraneopening is measured. This so-called closure time correlates with thefunctional efficiency of the platelets. A test cartridge for use in amethod for the determination of platelet function based on the closuretime is described, for example, in patent specification WO 97/34698.Thus far test cartridges that are equipped with a membrane that iscoated with collagen (Col) and also with either ADP or epinephrine (Epi)are used in the method for the determination of closure time. Differentpartition members as well as their preparation and use are described,for example in patent specification EP 716 744 B1.

Subject to the construction, a distinction is thus made between Col/ADPtest cartridges and Col/Epi test cartridges. Normally a patient sampleis first analyzed with the aid of a Col/Epi test cartridge. In the caseof an abnormally prolonged Col/Epi closure time, which indicates adisorder of platelet aggregation, a Col/ADP measurement is subsequentlycarried out. If the Col/ADP closure time is likewise abnormallyprolonged this is an indicator of platelet dysfunction or a disorder ofthe von Willebrand factor. If in contrast the Col/ADP closure time isnormal this can indicate the presence of acetylsalicylic acid or thepresence of an acquired or inherited thrombocytopathy such as, forexample, storage pool disease. A disadvantage of the PFA-100® system isthat the available Col/ADP and Col/Epi test cartridges have only alimited sensitivity for the aggregation inhibitory effect of plateletaggregation inhibitors of the thienopyridine group (e.g. clopidogrel,ticlopidine). A more reliable determination of the therapeutic effect ofthe medically used clopidogrel and ticlopidine, especially when thepatient has also taken ASA (e.g. Aspirin®) is hitherto not possible withthe help of the known Col/ADP and Col/Epi test cartridges in thePFA-100® system.

The patent specification WO 2005/007868 A2 describes an alternativemethod for the determination of platelet function that allows thedetection of the therapeutic effect of clopidogrel and other P2Y(12)antagonists. In this method a whole blood sample of a patient is mixedwith an anticoagulant and treated with ADP for the induction of plateletaggregation. In addition, prostaglandin E1 (PGE 1) is added to thesample. Prostaglandin E1, a product of human arachidonic acidmetabolism, is able to reduce the reactivity of platelets significantly,even in low doses, and is therefore also used for the inhibition ofplatelet activation. In the test method described in WO 2005/007868 A2,PGE 1 is used to reduce the undesirable activation of the ADP receptorP2Y(1) and thus to increase the specificity of the test method for theP2Y(12) receptor and for P2Y(12) antagonists such as clopidogrel. Inaddition, microparticles to which a ligand for the GPIIb/IIIa receptorsuch as, for example, fibrinogen is coupled, are added and theaggregation reaction is measured aggregometrically on the basis of theincreasing light transmission. A disadvantage of the previouslydescribed method is that as with light transmission aggregometryplatelet function is not determined under the influence of flowconditions and shear forces.

SUMMARY OF THE INVENTION

The object forming the basis of the present invention is to provide amethod for the determination of platelet function under flow conditionsthat allows the determination of the platelet aggregation inhibitoryeffect of P2Y(12) antagonists. The solution to the object lies in theprovision of the methods and materials according to the inventiondescribed in the claims.

The object of the present invention is an in vitro method for thedetermination of platelet function in a whole blood sample. Preferablythe whole blood sample is freshly drawn anticoagulated venous human oranimal blood that is to be investigated within four hours after bloodcollection with the help of the method according to the invention. Thewhole blood is preferably anticoagulated by the addition of ananticoagulant. Suitable for use as anticoagulant are bufferedcalcium-binding citrate solutions such as, for example, 3.2 or 3.8%buffered sodium citrate solutions, as well as natural or syntheticdirect thrombin inhibitors such as, for example, hirudin, PPACK(D-Phe-Pro-Arg-chloromethylketone, HCl), argatroban and melagatran, ornatural or synthetic direct Factor Xa inhibitors such as, for example,antistasin, tick anticoagulant peptide, yagin, draculin, GGACK(H-Glu-Glu-Arg-chloromethylketone), diamidino Factor Xa inhibitors andmonobenzamidine Factor Xa inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention for the determination of plateletfunction comprises several methodological steps. For the simulation ofthe physiological flow conditions prevailing in small arteries the bloodthat is initially located in a reservoir is passed though a capillarythat preferably has a diameter of about 200 μm. The capillary leads intoa measurement chamber that is separated into two compartments by apartition member. The partition member has an opening through which theblood is passed from the first into the second compartment. The methodof the invention is characterized in that the partition member usedcomprises an activator of purinergic receptors and an activator ofintracellular adenylate cyclases whereby the blood flowing through theopening of the partition member is brought into contact with thesesubstances contained in or on the partition member. As a result of theplatelet aggregation that is induced by the contact with the substancesa thrombus forms at the opening of the partition member. The time thatis necessary for the formation of the thrombus at the opening of thepartition member up to closure of the opening is measured. Preferablythe closure time is measured in that an apparatus is used that comprisesa pressure sensor which determines the blood flow through the apertureduring the test. Thus, after initial rapid aspiration of the dead volumeof the test cartridge the initial flow rate is first determined. If theflow rate falls below 10% of this initial flow rate for more than 3seconds the measurement is ended and the time passed until then isrecorded as the so-called closure time. This so-called closure time,which is i.a. dependent on the aggregation reaction of the stimulatedplatelets, is a measure of platelet function. Preferably the closuretime that was measured for a whole blood sample of a patient is comparedwith a closure time reference range for whole blood samples of healthysubjects.

Preferably the blood flow through the capillary and through the openingof the partition member is produced by creating a partial vacuum in themeasurement chamber, that is by suction. In a particularly preferredembodiment the partial vacuum is produced by the combined action of asuitable test cartridge and an apparatus. An example of such a system isdescribed, for example, in patent specification WO 97/034698.

The partition member used in the method according to the inventioncomprises an activator of purinergic receptors, preferably from thegroup adenosine-5′-diphosphate (ADP),2-methylthioadenosine-5′-diphosphate (2-MeSADP) and their derivatives.In a preferred embodiment a partition member is used that comprises anADP salt or a 2-MeSADP salt. In a preferred embodiment a partitionmember is used that comprises 1 to 100 μg, especially preferred 5 to 50μg, particularly preferred 20 to 25 μg ADP.

The partition member used comprises further an activator ofintracellular adenylate cyclases, preferably from the groupprostaglandin E1 (PGE 1), forskolin and its water-soluble derivatives,prostaglandin 12 and its stable derivatives, iloprost and cicaprost. Ina preferred embodiment a partition member is used that comprises 1 to1000 ng, especially preferably 3 to 20 ng prostaglandin E1. In anotherpreferred embodiment a partition member is used that comprises 0.1 to 10μg, especially preferred 0.5 to 5 μg forskolin.

In an especially preferred embodiment of the method a partition memberis used that comprises ADP and prostaglandin E1.

In a further preferred embodiment of the method according to theinvention a partition member is used that also comprises calcium ions,preferably in the form of calcium chloride dihydrate. In a preferredembodiment a partition member is used that comprises 50 to 200 μg,especially preferred 100 to 150 μg, most especially preferred 125 μgcalcium ions in the form of calcium chloride dihydrate.

It was found that in the presence of calcium ions the plateletaggregation inhibitory effect of acetylsalicylic acid (ASA) is reducedso considerably that an accurate determination of the plateletaggregation inhibitory (antithrombotic) effect of other plateletaggregation inhibitors such as, for example, P2Y(12) antagonists such asclopidogrel is also possible in such samples that comprise ASA. Apartition member that comprises calcium ions is thus especially then tobe used in the method according to the invention when the whole bloodsample to be investigated is anticoagulated with a calcium-bindinganticoagulant. If the whole blood sample to be investigated isanticoagulated with a non-calcium-binding anticoagulant such as, forexample, with a direct thrombin or Factor Xa inhibitor, the calcium ionconcentration contained endogenously in the sample is sufficient toreduce an ASA-induced platelet dysfunction. Nevertheless, a partitionmember that comprises calcium ions can also be used in these cases.

The method according to the invention is used most preferably for thedetermination of the antithrombotic (platelet aggregation inhibitory)effect of a P2Y(12) antagonist, especially for the determination of aP2Y(12) antagonist from the group clopidogrel, ticlopidine, prasugrel(synonym: CS-747) and other thienopyridines, AR-C67085MX(2-propylthio-D-β,γ-dichloromethylene-adenosine-5′-triphosphate),cangrelor (synonym: AR-C69931 MX,N6-[2-methylthio)ethyl]-2-(3,3,3-trifluoropropyl)thio-5′-adenylic acid),C1330-7(N1-(6-ethoxy-1,3-benzothiazol-2-yl-2-(7-ethoxy-4-hydroxy-2,2-dioxo-2H-2-6benzo[4,5][1,3]thiazole[2,3-c][1,2.4]thiadiazin-3-yl)-2-oxo-1-ethanesulfonamide),AZD 6140 (nucleoside analogs), MRS 2395 (2,2-dimethyl-propionic acid3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester) and 2-MeSAMP (2-methylthioadenosine-5′-monophospate).

Surprisingly, it was also found that the method according to theinvention can also be used for the determination of the antithrombotic(platelet aggregation inhibitory) effect of a P2Y(1) antagonist. Inparticular, the method can be used for the determination of theantithrombotic effect of P2Y(1) antagonists from the group MRS 2179[2′-deoxy-N-6-methyladenosine-3′,5′-bisphosphate, diammonium salt], MRS2279[(N)-methanocarba-N-6-methyl-2-chloro-2′deoxyadenosine-3′,5′-diphosphate],MRS 2500[2-iodo-N-6-methyl-(N)-methanocarba-2′-deoxyadenosine-3‘5’-diphosphate],A2P5P [adenosine-2′,5′-diphosphate], A3P5P[adenosine-3′,5′-diphosphate], A3P5PS[adenosine-3′-phosphate-5′-phosphosulfate].

A further object of the present invention concerns a device, as forexample a test cartridge, which is suitable for the determination ofplatelet function in a whole blood sample wherein the device comprisesdifferent elements: a) a reservoir for storing the sample; b) acapillary through which the blood is passed from the reservoir into ameasurement chamber; c) a measurement chamber that is separated into twocompartments by a partition member, wherein the first compartmentreceives the blood from the capillary; d) a partition member whichdivides the measurement chamber into two compartments and which has anopening through which the blood can flow from the first compartment intothe second compartment. The device is characterized in that thepartition member comprises an activator of purinergic receptors and anactivator of intracellular adenylate cyclases. In one preferredembodiment the partition member also comprises calcium ions, preferablyin the form of calcium chloride dihydrate.

The partition member is a porous or nonporous support matrix for anactivator of purinergic receptors and an activator of intracellularadenylate cyclases and optionally for calcium ions. Preferably thepartition member is constructed in the form of a membrane. The preferredmaterial is liquid absorbing so that the aforementioned substances canbe applied in solution. Especially preferred materials are celluloseesters, ceramic, nylon, polypropylene, polyether sulfone, andpolyvinylidene fluoride (PVDF). Preferably the partition member wettedor soaked with the desired substances is dried. By contact of the bloodwith the partition member the substances are dissolved from thepartition member and mix with the blood sample.

The partition member preferably has a circular opening that is producedin the support matrix by punching. The diameter of the opening in thepartition member is so dimensioned that a thrombus can form under theconditions of the respective method which closes the opening and canthus stop the blood flow. Preferably the opening in the partition memberhas a diameter between approximately 100 μm and approximately 200 μm.Particularly preferably the diameter of the opening in the partitionmember is about 100 μm.

The device according to the invention is preferably so constructed thata partial vacuum that brings about a blood flow from the reservoirthrough the capillary into the measurement chamber and through theopening of the partition member is produced in the device with the helpof an apparatus that is integrated with components of the device.

The present invention further relates to the use of a device accordingto the invention in a method for the determination of platelet function.A preferred use of a device according to the invention relates to theuse for the determination of the antithrombotic effect of a P2Y(12)antagonist. Another preferred use of a device according to the inventionrelates to the use for the determination of the antithrombotic effect ofa P2Y(1) antagonist.

The following embodiment examples serve to illustrate the methodaccording to the invention and are not to be understood as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

FIG. 1 shows by way of example how a device for the determination ofplatelet function according to the invention can be constructed. Shownis a test cartridge in accordance with WO 97/34698 in longitudinalsection that is placed in a suitable apparatus for implementing themethod according to the invention and into which extends a vacuumapparatus (15) that is responsible for the generation of the partialvacuum The vacuum apparatus (15) has a ring gasket (27) which is locatedas a seal on the circumferential edge (12) of the sample container (10).The test cartridge has a housing that forms a reservoir (61) and a testchamber (63). The test chamber (63) is constructed to accept a samplecontainer (10) the cavity of which can also be referred to asmeasurement chamber. The sample container (10) supports a partitionmember (6) treated with reagents and with a central opening (aperture)and a capillary attachment (30, 31) that connects the capillary (40)with the sample container (10). Reservoir (61) and test chamber (63) areseparated by a penetrable element (70). The figure shows a phase of thetest cycle after the vacuum apparatus (15) is in contact with samplecontainer (10) and has moved downwards so that the base of the samplecontainer (10) is in contact with the support (71) and the capillary(40) has penetrated the penetrable element (70) and penetrated into thesample (11). The apparatus produces a partial vacuum in the samplecontainer (10) by means of which the sample (11) is pulled through thecapillary (40) into the first compartment (18) of the measurementchamber and then through the opening in the partition member (6).

FIG. 2

Diagram for the illustration of closure times (in seconds [s]) fornormal untreated whole blood samples (control) and for whole bloodsamples that had been treated with the P2Y(12) antagonist MRS 2395and/or the COX-1 inhibitor acetylsalicylic acid (ASA) in vitro (seeExample 2). Whole blood samples from 11 healthy donors anticoagulatedwith sodium citrate were used. On the left of the diagram are shown themean values and the standard deviations of the closure times determinedwith the ADP/PGE1/calcium test cartridge according to the invention(cut-off: 81 seconds). On the right of the diagram are shown the meanvalues and the standard deviations of the closure times that weredetermined for comparison with conventional Col/Epi test cartridges(cut-off: 158 seconds). A comparison of the two types of test cartridgeshows that with use of an ADP/PGE1 test cartridge according to theinvention, closure times that lie significantly above the upperreference value (cut-off) were measured with samples that were treatedwith the P2Y(12) antagonist MRS 2395, whereas the same samples with theuse of a Col/Epi test cartridge lie to a greater extent below the upperreference value (cut-off). That means that the method for determinationof platelet function according to the invention allows a more sensitivedetermination of platelet dysfunction induced by a P2Y(12) antagonistthan the comparison method from the prior art.

FIG. 3

Diagram for illustration of the closure times (in seconds [s]) fornormal untreated whole blood samples (control) and for whole bloodsamples treated in vitro with the P2Y(12) antagonist MRS 2395, theP2Y(1) antagonist MRS 2179 or the COX-1 inhibitor acetylsalicylic acid(ASA) (see example 4). Whole blood samples from 10 healthy donorsanticoagulated with PPACK were used. In the diagram are shown the meanvalues and the standard deviations of the closure times that weredetermined with the ADP/PGE1 test cartridges according to the invention(cut-off: 90 seconds). The performance evaluation shows that with use ofan ADP/PGE1 test cartridge according to the invention, closure timesthat lie significantly above the reference value (cut-off) were measuredwith samples that were treated with the P2Y(12) antagonist MRS 2395 orthe P2Y(1) antagonist MRS 2179, whereas the samples treated with COX-1inhibitor acetylsalicylic acid show no prolongation of closure times andthus lie below the cut-off.

EXAMPLES Example 1 Preparation of an ADP/PGE1/Calcium Test CartridgeAccording to the Invention

For the preparation of a partition member for a test cartridge accordingto the invention a polyether sulfone filter membrane (Supor®) membrane,Pall GmbH, Dreieich, Germany) was cut into strips. 1 μL of a solutioncomprising 7 μg/μL ADP (adenosine-5′-diphosphate potassium salt.2H₂O,Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 5 ng/μL PGE1(prostaglandin E1, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and367.5 μg/μL CaCl₂.2H₂O (equivalent to 100 μg/μL Ca²⁺ ions) were pipettedpunctiform onto the membrane and the membrane was dried. Next a circularopening (aperture) with a diameter of 100 μm was punched out of themiddle of the region of the membrane treated with the reagents. Themembrane thus prepared was used as partition member in the measurementchamber of a PFA-100® test cartridge (Dade Behring Marburg GmbH,Marburg, Germany).

Example 2 Use of an ADP/PGE1/Calcium Test Cartridge According to theInvention for the Determination of the Antithrombotic Effect of aP2Y(12) Antagonist in Vitro

2a) Sample Preparation

Venous blood was taken from 11 healthy donors and anticoagulated withsodium citrate (3.2% buffered Na citrate).

Aliquots of the citrated whole blood sample were treated in vitro withthe P2Y(12) antagonist MRS 2395 (Sigma-Aldrich Chemie GmbH, Steinheim,Germany). For this purpose an ethanolic MRS 2395 stock solution (15mg/mL) was mixed with the whole blood samples so that an endconcentration of 100 μmol/L was obtained.

Further aliquots of the citrated whole blood samples were treated invitro with the COX-1 inhibitor acetylsalicylic acid (abbr.: ASA;Sigma-Aldrich Chemie GmbH, Steinheim, Germany). For this purpose anaqueous ASA stock solution (1 mg/mL) was mixed with whole blood samplesso that an end concentration of 30 μmol/L was obtained.

Further aliquots of the citrated whole blood samples were treated invitro with MRS 2395 and with ASA so that the previously stated endconcentrations were achieved.

After addition of the reagents the blood samples were incubated at roomtemperature for 5 minutes.

2b) Determination of the Antithrombotic Effect of MRS 2395 by ADPInduced Light Transmission Aggregometry (According to Born)

In order to check whether the samples treated with MRS 2395 actuallyshow a reduced platelet aggregation, platelet rich (PRP) and plateletpoor (PPP) plasma was prepared from aliquots of the untreated and MRS2395-treated whole blood samples described under Example 2a), and thesamples were then treated with 2 μM ADP. The PPP samples were used asblank controls. The photometric measurement of the aggregation reactionwas carried out in the automated coagulation apparatus BCT® (DadeBehring Marburg GmbH, Marburg, Germany) under continuous stirring (600rpm). The platelet aggregation of the samples treated with MRS 2395 wasreduced by a mean of 27% compared with the platelet aggregation of theuntreated samples.

2c) Determination of the Antithrombotic Effect of MRS 2395 by the MethodAccording to the Invention Under Flow Conditions

To determine the closure time as a measure of platelet function thewhole blood samples described under Example 2a) were investigated withthe aid of the ADP/PGE1/calcium test cartridge according to theinvention described in Example 1 in a PFA-100® apparatus (PlateletFunction Analyzer-100, Dade Behring Marburg GmbH, Marburg, Germany). Forthis purpose 700 μL of a blood sample were placed in the reservoir ofthe temperature equilibrated test cartridge (+37° C.) and incubated at+37° C. for 3 minutes. Next a partial vacuum of −40 mbar was generatedby the apparatus by which means the blood was sucked through a capillaryfrom the reservoir (diameter 200 μm) and finally through an opening(aperture) of the partition member in the measurement chamber. The timerequired up to the closure of the aperture by formation of a blood clotwas determined as closure time. Every sample investigated was determinedin duplicate and the mean value of a duplicate determination was used asthe measurement value.

For comparison purposes the whole blood samples described under Example2a) were investigated in parallel with a known Col/Epi PFA-100® testcartridge (2 μg collagen and 10 μg epinephrine on the membrane; 150 μmaperture diameter; Dade Behring Marburg GmbH, Marburg, Germany) in thePFA-100® apparatus.

The results of the investigations are summarized in FIG. 2 and therelevant figure description.

In Table 1 details are reported for how many of the respective 11 MRS2395- and/or acetylsalicylic acid-treated samples a closure time abovethe cut-off was measured with the aid of the ADP/PGE1 test cartridgeaccording to the invention and with the conventional Col/Epi testcartridge. In 9 of the 11 samples treated with MRS 2395 an abnormallyreduced platelet aggregation was measured with the aid of the methodaccording to the invention, whereas only 4 of 11 samples were classifiedas abnormal with the aid of the conventional method. That means that themethod according to the invention has an increased sensitivity for aplatelet dysfunction induced by a P2Y(12) antagonist. Moreover, it is ofadvantage that in the presence of free calcium ions the method accordingto the invention has a very low sensitivity for acetylsalicylic acid. Inonly one of 11 acetylsalicylic acid-treated samples, an abnormallyreduced platelet aggregation is measured with the aid of the methodaccording to the invention, whereas, in contrast, with the conventionalCol/Epi test cartridge 8 of the 11 samples treated with acetylsalicylicacid were determined as abnormal. Samples that are treated with MRS 2395and acetylsalicylic acid are classified 100% as abnormal with the aid ofthe conventional method, whereas only 9 of the 11 samples (as with soleaddition of MRS 2395) are classified as abnormal with the methodaccording to the invention. Thus on the basis of its high sensitivityfor platelet dysfunction induced by P2Y(12) antagonists and its lowsensitivity to platelet dysfunction induced by acetylsalicylic acid, themethod according to the invention is suitable for differentiation of thetwo classes of antithrombotics.

TABLE 1 Number of samples with closure times above cut-off (n = 11)Sample MRS 2395 MRS 2395 + (P2Y(12) Acetylsalicylic acid AcetylsalicylicTest cartridge antagonist) (COX-1 inhibitor) acid ADP/PGE1 9 1 9 Col/Epi4 8 112d) Determination of the Reference Range for Col/Epi and ADP/PGE1 TestCartridges

Venous blood was taken from healthy donors and anticoagulated withsodium citrate (3.2% buffered Na citrate). The closure timedetermination was carried out for each whole blood sample in thePFA-100® apparatus. Samples from 186 donors were determined in duplicatewith a Col/Epi PFA-100® test cartridge [see Example 2c)]. Samples from159 donors were determined in duplicate with an ADP/PGE1/calcium testcartridge according to the invention [see Examples 1 and 2c)].

The reference ranges (normal range) for the Col/Epi closure time and theADP/PGE1 closure time were established in that the measurement valueranges in which 90% of the measurement values found for the healthysubjects lay were determined (90% central interval of the normaldistribution of all measurements). This gave the following referenceranges for the closure times:

Col/Epi 70–158 seconds ADP/PGE1  46–81 seconds.

The upper reference limit of the reference range was defined as cut-off,i.e. as threshold value, for a platelet dysfunction. If the closure timeof a patient sample deviates from the reference range it can indicate aplatelet dysfunction. This means Col/Epi closure times that are greaterthan 158 seconds and ADP/PGE1 closure times that are greater than 81seconds indicate the presence of a platelet dysfunction within the senseof a reduced aggregation efficiency.

Example 3 Use of an ADP/PGE1 Test Cartridge According to the Inventionfor the Determination of the Antithrombotic Effect of the P2Y(12)Antagonist Clopidogrel ex Vivo

Venous blood was taken from 13 patients suffering from peripheralarterial obstructive disease and who had been treated with a daily doseof 75 mg clopidogrel (Plavix®, Sanofi-Aventis) as sole antithromboticfor a period of at least 4 weeks and the blood was anticoagulated withsodium citrate (3.8% buffered Na citrate). The samples were investigatedwith the aid of different methods to determine platelet function.

-   1. according to the invention under flow conditions and use of an    ADP/PGE1/calcium PFA-100® test cartridge (see Examples 1 and 2c),    cut-off: >81 seconds;-   2. under flow conditions and use of a PFA-100® test cartridge that    differed from the test cartridge according to the invention in    accordance with Example 1 in that it comprised no PGE1, cut-off: >78    seconds;-   3. with ADP-induced light transmission aggregometry (according to    Born) with addition of 2 μM ADP (see Example 2b), cut-off: <40%    light transmission at end of test;-   4. with ADP-induced light transmission aggregometry (according to    Born) with the addition of 5 μM ADP (see example 2b), cut-off: <40%    light transmission at end of test. This method is recommended by    Sanofi-Aventis, the manufacturer of the clopidogrel preparation    Plavix®, for the determination of the antithrombotic effect of the    medicament.

The cut-offs for the individual test procedures were determined bypreliminary investigations with whole blood and plasma samples of normaldonors.

Table 2 presents in detail with which of the four methods in which ofthe 13 patient samples an antithrombotic effect of clopidogrel could bedetected. “+” means an antithrombotic effect could be detected. “−”means no antithrombotic effect could be detected. “0” means that theduplicate determination gave contradictory results, i.e. one value aboveand one value below the cut-off.

TABLE 2 Method 1 2 3 4 5 6 7 8 9 10 11 12 13 Sensitivity 1.) ADP/PGE1 −0 + + + − + + + + + + + 77% 2.) ADP − − + + + − − − + − + − + 46% 3.)Aggregometry − + − + + − − + + + + + + 69%    2 μM ADP 4.) Aggregometry− − − + + − − + + + + + + 62%    5 μM ADP

With the aid of the method of the invention the antithrombotic effect ofclopidogrel intake could be detected in 10 of the 13 patients (77%). Theduplicate determination gave contradictory results with one patient(patient no. 2), whilst with 2 patients (patient no. 1 and 6) no reducedplatelet aggregation could be detected. However, in these two patientsan effect of dosage of clopidogrel could not be detected in any of themethods used.

The method according to the invention is more sensitive towards theplatelet dysfunction induced by clopidogrel than the standard method ofADP-induced light transmission aggregometry according to Born and moresensitive than the method with which a test cartridge is used thatcomprises ADP but no PGE1.

Example 4 Use of an ADP/PGE1 Test Cartridge According to the Inventionfor the Determination of the Antithrombotic Effect of a P2Y(12) and aP2Y(1) Antagonist in Vitro

4a) Sample Preparation

Venous blood was taken from 10 healthy donors and anticoagulated with 75μM PPACK.

Aliquots of the whole blood samples were treated in vitro with theP2Y(12) antagonist MRS 2395 (Sigma-Aldrich Chemie GmbH, Steinheim,Germany). For this purpose an ethanolic MRS 2395 stock solution (15mg/mL) was mixed with the whole blood samples so that an endconcentration of 150 μmol/L was obtained.

Further aliquots of the whole blood samples were treated in vitro withthe COX-1 inhibitor acetylsalicylic acid (abbr. ASA; Sigma-AldrichChemie GmbH, Steinheim, Germany). For this purpose an aqueous ASA stocksolution (1 mg/mL) was mixed with the whole blood samples so that an endconcentration of 30 μmol/L was obtained.

Further aliquots of the whole blood samples were treated in vitro withthe P2Y(1) antagonist MRS 2179 (Sigma-Aldrich Chemie GmbH, Steinheim,Germany). For this purpose an aqueous MRS 2179 stock solution (1 mg/mL)was mixed with the whole blood samples so that an end concentration of75 mol/L was obtained.

After addition of the reagents the blood samples were incubated at roomtemperature for 5 minutes.

4b) Determination of the Antithrombotic Effect of MRS 2395 and MRS 2179with the Aid of the Method According to the Invention Under FlowConditions

For the determination of the closure time as a measure of the plateletfunction the whole blood samples described under Example 4a) wereinvestigated with the aid of a ADP/PGE1 test cartridge according to theinvention in a PFA-100® apparatus (Platelet Function Analyzer-100, DadeBehring Marburg GmbH, Marburg, Germany). The ADP/PGE1 test cartridgeaccording to the invention used was prepared essentially as described inExample 1 but without the partition member having been treated withCaCl₂.2H₂O. The test cartridge thus comprised 7 μg ADP and 5 ng PGE1 butno calcium ions.

700 μL of a blood sample was added to the reservoir of the temperatureequilibrated test cartridge (+37° C.) and incubated at +37° C. in theapparatus for 3 minutes. Next a partial vacuum of −40 mbar was appliedby the apparatus when the blood was sucked through a capillary (diameter200 μm) from the reservoir and finally through an opening (aperture) ofthe partition member in the measurement chamber. The time that wasrequired up to the closure of the aperture by formation of a blood clotwas determined as closure time. Every sample investigated was determinedin duplicate and the mean value of a duplicate determination was used asthe measurement value.

The results of the investigations are summarized in FIG. 3 and therelevant figure description.

In Table 3 details are given for how many of the respective 10 MRS2395-, MRS 2179- or acetylsalicylic acid-treated samples a closure timeabove the cut-off was measured with the aid of the ADP/PGE1 testcartridge according to the invention. In 9 of the 10 samples treatedwith MRS 2395 and in 10 of the 10 samples treated with MRS 2179 anabnormally reduced platelet aggregation was measured with the aid of themethod of the invention, whereas none of the samples treated withacetylsalicylic acid was classified as abnormal. This means that themethod according to the invention has a high sensitivity for a plateletdysfunction induced both by a P2Y(12) antagonist and by a P2Y(1)antagonist. Moreover, it is of advantage that the method according tothe invention has a very low sensitivity for acetylsalicylic acid in thepresence of free calcium ions.

Thus on the basis of its high sensitivity for platelet dysfunctioninduced by ADP receptor antagonists and its low sensitivity towardsplatelet dysfunction induced by acetylsalicylic acid the methodaccording to the invention is suitable for differentiation of the twoclasses of antithrombotics.

TABLE 3 Number of samples with closure times above cut-off (n = 10)Sample MRS 2395 Acetylsalicylic MRS 2179 (P2Y(12) acid (P2Y(1) Testcartridge antagonist) (COX-1 inhibitor) antagonist) ADP/PGE1 9 0 104c) Determination of the Cut-Off for ADP/PGE Test Cartridges

Owing to the use of PPACK as anticoagulant the cut-off determined withcitrated whole blood in the above-described examples cannot be used.Therefore, the reference range for the ADP/PGE1 closure time from the 10samples of the healthy donors treated with PPACK was calculated by thedetermination of the 90% central interval of the normal distribution ofthe mean values of the duplicate determinations. This gave the followingreference ranges for the closure times:

ADP/PGE1 51-90 seconds.

The upper limit of the 90% central interval was defined as cut-off, i.e.as threshold value for a platelet dysfunction.

1. A method for the determination of platelet function in a whole bloodsample, the method comprising the following steps: a) passing the bloodthrough a capillary and then through an opening of a partition member;and b) measuring the time that is required for the formation of athrombus at the opening of the partition member up to closure of theopening; wherein the partition member comprises: i) an activator ofpurinergic receptors and ii) an activator of intracellular adenylatecyclases.
 2. The method as claimed in claim 1, wherein the partitionmember comprises in addition calcium ions.
 3. The method as claimed inclaim 1 wherein the partition member used comprises an activator ofpurinergic receptors from the group adenosine-5′-diphosphate,2-methylthioadenosine-5′-diphosphate, and their derivatives.
 4. Themethod as claimed in claim 1, wherein the partition member comprises anactivator of intracellular adenylate cyclases from the groupprostaglandin E1, forskolin and their derivatives, prostaglandin I2 andits stable derivatives, illoprost, and cicaprost.
 5. The method asclaimed in claim 1, wherein the whole blood sample is anticoagulatedwith a direct thrombin inhibitor.
 6. The method as claimed in claim 1,wherein the whole blood sample is anticoagulated with a direct Factor Xainhibitor.
 7. The method as claimed in claim 2, wherein the whole bloodsample is anticoagulated with citrate.
 8. The method as claimed in claim1 wherein the method determines the antithrombotic effect of a P2Y(12)antagonist.
 9. The method as claimed in claim 8 wherein the P2Y(12)antagonist is selected from clopidogrel, ticlopidine, prasugrel,AR-C67085MX, cangrelor, C1330-7, MRS 2395, and2-methylthioadenosine-5′-monophosphate.
 10. The method as claimed inclaim 1 wherein the method determines the antithrombotic effect of aP2Y(1) antagonist.
 11. The method as claimed in claim 10 wherein theP2Y(1) antagonist is selected from MRS 2179, MRS 2279, MRS 2500, A2P5P,A3P5P, and A3P5PS.
 12. A device for the determination of plateletfunction in a whole blood sample wherein the device comprises thefollowing elements: a) a reservoir for holding the sample; b) acapillary through which the blood is passed from the reservoir into ameasurement chamber; c) a measurement chamber that is divided into twocompartments by a partition member, wherein the first compartmentreceives the blood from the capillary; d) a partition member thatdivides the measurement chamber into two compartments and has an openingthrough which the blood can flow from the first compartment into thesecond compartment; wherein the partition member comprises i) anactivator of purinergic receptors and ii) an activator of intracellularadenylate cyclases.
 13. The device as claimed in claim 12, wherein thepartition member also comprises calcium ions.
 14. A method for thedetermination of platelet function in a whole blood sample, comprisingpassing the blood sample through the device as claimed in claim
 12. 15.The method as claimed in claim 14 wherein the method determines theantithrombotic effect of a P2Y(12) antagonist.
 16. The method as claimedin claim 14 wherein the method determines the antithrombotic effect of aP2Y(1) antagonist.